Helper-free stocks of recombinant adeno-associated virus vectors

ABSTRACT

The present invention relates to a method for producing helper-free stocks of recombinant adeno-associated virus (rAAV) which can be used to efficiently and stably transduce foreign genes into host cells or organisms. The method comprises the cotransfection of eukaryotic cells with rAAV and with helper AAV DNA in the presence of helper virus (e.g. adenovirus or herpesvirus) such that the helper AAV DNA is not associated with virion formation. The crux of the invention lies in the inability of the helper AAV DNA to recombine with rAAV vector, thereby preventing the generation of wild-type virus. In a specific embodiment of the invention, the vector comprises a recombinant AAV genome containing only the terminal regions of the AAV chromosome bracketing a non-viral gene, and the helper AAV DNA comprises a recombinant AAV genome containing that part of the AAV genome which is not present in the vector, and in which the AAV terminal regions are replaced by adenovirus sequences. In a further embodiment of the invention, cell lines are created which incorporate helper AAV DNA which can directly produce substantially pure recombinant AAV virus. The pure stocks of recombinant AAV produced according to the invention provide an AAV viral expression vector system with increased yield of recombinant virus, improved efficiency, higher definition, and greater safety than presently used systems.

1. FIELD OF THE INVENTION

The present invention relates to a method for producing substantiallypure stocks of recombinant adeno-associated virus (AAV), free of theadeno-associated helper virus found associated with previously availablerecombinant AAV. According to the invention, the substantially purestocks of recombinant AAV may be used to introduce exogenous geneticsequences into cells, cell lines, or organisms; in the absence of theadeno-associated helper virus, the recombinant AAV will remain stablyintegrated into cellular DNA. In another embodiment of the inventioncells containing integrated recombinant AAV may be exposed to helperviruses, resulting in excision, replication, and amplification ofintegrated sequences, thereby providing a means for achieving increasedexpression of gene product. The present invention also provides fornovel recombinant AAV vectors and adeno-associated helper viruses.

2. BACKGROUND OF THE INVENTION 2.1. Viral Vectors

Viral vectors permit the expression of exogenous genes in eukaryoticcells, and thereby enable the production of proteins which requirepostranslational modifications unique to animal cells. Viral expressionvectors (reviewed in Rigby, 1983, J. Gen. Virol. 64:255-266) have beendeveloped using DNA viruses, such as papovaviruses (i.e. SV40),adenoviruses, herpes viruses, and poxviruses (i.e. vaccinia virus,Mackett et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419;Panicoli et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4927-4931) andRNA viruses, such as retroviruses.

In disclosing the construction and applications of a murine retrovirusshuttle vector, Cepko et al. (1984, Cell 37:1053-1062) cites severalproperties which may be desirable in a mammalian gene transfer system,including the ability of the vector to be introduced into a wide rangeof hosts and the recoverability of transferred sequences as molecularclones (i.e. a vector which can “shuttle” between animal and bacterialcells; see DiMaio et al., 1982, Proc. Natl. Acad. Sci. U.S.A.79:4030-4034). As efficient shuttle vectors, retroviruses have become apopular vehicle for transferring genes into eukaryotic cells. Retroviruspackaging cell lines (Mann et al., 1983, Cell 33:153-159; Watanabe andTemin, 1983, Mol. Cell. Biol. 3:2241-2249; Cohn and Mulligan, 1984,Proc. Natl. Acad. Sci. U.S.A. 81:6349-6353; Sorge et al., 1984, Mol.Cell. Biol. 4:1730-1737) allow production of replication-defectiveretrovirus vectors in the absence of helper virus; the defectiveretroviral vectors are able to infect and integrate into cells butcannot replicate. The ability to produce helper-free stocks of defectiveretroviruses using packaging cell lines protects against spread of therecombinant virus, and avoids possible dissemination of recombinantvirus-induced disease. However, some retrovirus packaging lines havebeen shown to produce only low titers of retroviral vectors, or producehigh levels of helper virus; furthermore, some retroviruses exhibitlimited host ranges (Miller and Baltimore, 1986, Mol. Cell. Biol.6:2895-2902). The recognition of human retroviruses over the past decadeas the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS) andsome cases of T-cell and hairy cell leukemia, and the numerous examplesof oncogenic animal retroviruses, have created an awareness of healthrisks potentially associated with the use of retrovirus vectors,particularly relevant to future prospects in human gene therapy. Many ofthe alternative viral vectors currently available either do notintegrate into host cells at high frequency, are not easily rescuablefrom the integrated state, are limited in their host range, or includeother viral genes, thereby creating a need for the development of a safeand efficient viral vector system.

2.2. Adeno-Associated Virus

Adeno-associated virus (AAV) is a defective member of the parvovirusfamily. The AAV genome is encapsidated as a single-stranded DNA moleculeof plus or minus polarity (Berns and Rose, 1970, J. Virol. 5:693-699;Blacklow et al., 1967, J. Exp. Med. 115:755-763). Strands of bothpolarities are packaged, but in separate virus particles (Berns andAdler, 1972, Virology 9:394-396) and both strands are infectious(Samulski et al., 1987, J. Virol. 61:3096-3101).

The single-stranded DNA genome of the human adeno-associated virus type2 (AAV2) is 4681 base pairs in length and is flanked by invertedterminal repeated sequences of 145 base pairs each (Lusby et al., 1982,J. Virol. 41:518-526). The first 125 nucleotides form a palindromicsequence that can fold back on itself to form a “T”-shaped hairpinstructure and can exist in either of two orientations (flip or flop),leading to the suggestion (Berns and Hauswirth, 1979, Adv. Virus Res.25:407-449) that AAV may replicate according to a model first proposedby Cavalier-Smith for linear-chromosomal DNA (1974, Nature 250:467-470)in which the terminal hairpin of AAV is used as a primer for theinitiation of DNA replication. The AAV sequences that are required incis for packaging, integration/rescue, and replication of viral DNAappear to be located within a 284 base pair (bp) sequence that includesthe terminal repeated sequence (McLaughlin et al., 1988, J. Virol.62:1963-1973).

At least three regions which, when mutated, give rise to phenotypicallydistinct viruses have been identified in the AAV genome (Hermonat etal., 1984, J. Virol. 51:329-339). The rep region codes for at least fourproteins (Mendelson et al., 1986, J. Virol. 60:823-832) that arerequired for DNA replication and for rescue from the recombinantplasmid. The cap and lip regions appear to encode for AAV capsidproteins; mutants containing lesions within these regions are capable ofDNA replication (Hermonat et al., 1984, J. Virol. 51:329-339). AAVcontains three transcriptional promoters (Carter et al., 1983, in “TheParvoviruses”, K. Berns ed., Plenum Publishing Corp., NY pp. 153-207;Green and Roeder, 1980, Cell 22:231-242; Laughlin et al., 1979, Proc.Natl. Acad. Sci. U.S.A. 76:5567-5571; Lusby and Berns, 1982, J. Virol.41:518-526; Marcus et al., 1981, Eur. J. Biochem. 121:147-154). Theviral DNA sequence displays two major open reading frames, one in theleft half and the other in the right half of the conventional AAV map(Srivastava et al., 1985, J. Virol. 45:555-564).

AAV-2 can be propagated as a lytic virus or maintained as a provirus,integrated into host cell DNA (Cukor et al., 1984, in “TheParvoviruses,” Berns, ed., Plenum Publishing Corp., NY pp. 33-66).Although under certain conditions AAV can replicate in the absence ofhelper virus (Yakobson et al., 1987, J. Virol. 61:972-981), efficientreplication requires coinfection with either adenovirus (Atchinson etal., 1965, Science 194:754-756; Hoggan, 1965, Fed. Proc. Am. Soc. Exp.Biol. 24:248; Parks et al., 1967, J. Virol. 1:171-180); herpes simplexvirus (Buller et al., 1981, J. Virol. 40:241-247) or cytomegalovirus,Epstein-Barr virus, or vaccinia virus. Hence the classification of AAVas a “defective” virus.

When no helper virus is available, AAV can persist in the host cellgenomic DNA as an integrated provirus (Berns et al., 1975, Virology68:556-560; Cheung et al., 1980, J. Virol. 33:739-748). Virusintegration appears to have no apparent effect on cell growth ormorphology (Handa et al., 1977, Virology 82:84-92; Hoggan et al., 1972,in “Proceedings of the Fourth Lepetit Colloquium, North HollandPublishing Co., Amsterdam pp. 243-249). Studies of the physicalstructure of integrated AAV genomes (Cheung et al., 1980, supra; Bernset al., 1982, in “Virus Persistence”, Mahy et al., eds., CambridgeUniversity Press, NY pp. 249-265) suggest that viral insertion occurs atrandom positions in the host chromosome but at a unique position withrespect to AAV DNA, occurring within the terminal repeated sequence.Integrated AAV genomes have been found to be essentially stable,persisting in tissue culture for greater than 100 passages (Cheung etal., 1980 supra).

Although AAV is believed to be a human virus, its host range for lyticgrowth is unusually broad. Virtually every mammalian cell line(including a variety of human, simian, and rodent cell lines) evaluatedcould be productively infected with AAV, provided that an appropriatehelper virus was used (Cukor et. al., 1984, in “The Parvoviruses”,Berns, ed. Plenum Publishing Corp., NY, pp. 33-66).

No disease has been associated with AAV in either human or animalpopulations (Ostrove et al., 1987, Virology 113:521-533) despitewidespread exposure and apparent infection. Anti-AAV antibodies havebeen frequently found in humans and monkeys. It is estimated that about70 to 80 percent of children acquire antibodies to AAV types 1, 2, and 3within the first-decade; more than 50 percent of adults have been foundto maintain detectable anti-AAV antibodies. AAV has been isolated fromfecal, ocular, and respiratory specimens during acute adenovirusinfections, but not during other illnesses (Dulbecco and Ginsberg, 1980,in “Virology”, reprinted from Davis, Dulbecco, Eisen and Ginsberg's“Microbiology”, Third Edition, Harper and Row Publishers, Hagerstown, p.1059).

2.3. Recombinant Adeno-Associated Virus

Samulski et al., (1982, Proc. Natl. Acad. Sci. U.S.A. 79:2077-2081)cloned intact duplex AAV DNA into the bacterial plasmid pBR322 and foundthat the AAV genome could be rescued from the recombinant plasmid bytransfection of the plasmid DNA into human cells with adenovirus 5 ashelper. The efficiency of rescue from the plasmid was sufficiently highto produce yields of AAV DNA comparable to those observed aftertransfection with equal amounts of purified virion DNA.

The AAV sequences in the recombinant plasmid could be modified, and then“shuttled” into eukaryotic cells by transfection. In the presence ofhelper adenovirus, the AAV genome was found to be rescued free of anyplasmid DNA sequences and replicated to produce infectious AAV particles(Samulski et al., 1982, Proc. Natl. Acad. Sci. 79:2077-2081; Laughlin etal., 1983, Gene 23:65-73; Samulski et al., 1983, Cell 33:135-143;Senapathy et al., 1984, J. Mol. Biol. 179:1-20).

The AAV vector system has been used to express a variety of genes ineukaryotic cells. Hermonat and Muzyczka (1984, Proc. Natl. Acad. Sci.U.S.A. 81:6466-6470) produced a recombinant AAV (rAAV) viral stock inwhich the neomycin resistance gene (neo). was substituted for AAV capsidgene and observed rAAV transduction of neomycin resistance into murineand human cell lines. Tratschen et al. (1984), Mol. Cell. Biol.4:2072-2081) created a rAAV which was found to express thechloramphenicol acetyltransferase (CAT) gene in human cells. Lafare etal. (1988, Virology 162:483-486) observed gene transfer intohematopoietic progenitor cells using an AAV vector. Ohi et al. (1988, J.Cell. Biol. 107:304A) constructed a recombinant AAV genome containinghuman β-globin cDNA. Wondisford et al. (1988, Mol. Endocrinol. 2:32-39)cotransfected cells with two different recombinant AAV vectors, eachencoding a subunit of human thyrotropin, and observed expression ofbiologically active thyrotropin.

Several AAV vector systems have been designed. Samulski et al. (1987, J.Virol. 61:3096-3101) constructed an infectious adeno-associated viralgenome that contains two XbaI cleavage sites flanking the viral codingdomain; these restriction enzyme cleavage sites were created to allownonviral sequences to be inserted between the cis-acting terminalrepeats of AAV.

U.S. Pat. No. 4,797,368, Carter and Tratschen, filed May 15, 1985,issued Jan. 10, 1989 relates to AAV vectors contained in a plasmid,capable of being packaged into AAV particles, and functioning as avector for stable maintenance or expression of a gene or a DNA sequencein eukaryotic cells when under control of an AAV transcription promoter.

A problem encountered in all AAV systems prior to the present inventionhas been the inability to produce recombinant virus stocks free ofhelper AAV virus. The presence of helper AAV virus can potentiallyresult in continued spread of recombinant AAV, could detract from theefficiency of rAAV production and the transduction of foreign genes, andcould interfere with efficient expression of the foreign genes. Further,recombinant virus stocks produced using prior AAV helper systems did notproduce a linear increase in the number of cells containing stabilyintegrated recombinant virus DNA as the multiplicity of infectionincreased. This presumably resulted, at least in part, from inhibitoryeffects of AAV gene products expressed by helper AAV virus.

Various methods have been used in attempts to decrease the percentage ofcontaminating helper virus. Hormonat and Muzyczka (1984, supra) insertedbacteriophage λ sequences into a nonessential region of rAAV whichresulted in a DNA length too large to package into virions; a variablenumber of virions containing wild-type AAV continued to contaminate rAAVstocks. Lebkowski et al. (1988, Mol. Cell. Biol. 3:3988-3996) report amethod for producing recombinant AAV stocks with minimal contaminationby wild-type virus, in which deletion mutant AAV are used to complementrecombinant AAV viral functions. According .to the method of Lebkowskiet al., two independent recombinant events were required to producewild-type contaminants. However, given the large number of viralparticles produced during productive infection, a significant number ofwild-type virus were generated which contaminated the recombinant stock.U.S. Pat. No. 4,797,368 uses various methods including using rep(−)(replication deficient) helper virus, to reduce the level ofcontaminating wild-type AAV, but acknowledge that “it is not as yetpossible to completely avoid generation of wild-type recombinants.”

3. SUMMARY OF THE INVENTION

The present invention relates to a method for producing substantiallyhelper-free stocks of recombinant adeno-associated virus (rAAV) whichcan be used to efficiently and stably transduce foreign genes into hostcells or organisms. The method comprises the cotransfection ofeukaryotic cells with rAAV and with helper AAV DNA in the presence ofhelper virus (e.g. adenovirus or herpesvirus) such that the helper AAVDNA is not associated with virion formation. The crux of the inventionlies in the inability of the helper AAV DNA to recombine with rAAV,thereby preventing the generation of wild-type virus.

In a specific embodiment of the invention, the vector comprises arecombinant AAV genome containing only the terminal regions of the AAVchromosome bracketing a non-viral gene, and the helper AAV DNA comprisesa recombinant AAV genome containing that part of the AAV genome which isnot present in the vector, and in which the AAV terminal regions arereplaced by adenovirus terminal sequences. The substantially pure stocksof recombinant AAV produced according to the invention provide an AAVviral expression vector system with efficient yield of helper-freerecombinant virus. These stocks are able to introduce a foreign geneinto a recipient cell at higher efficiency than has been obtainedpreviously using stocks that contain helper AAV virus.

In a further embodiment of the invention, the helper AAV virus DNA maybe incorporated into a cell line, such that rAAV constructs may be growndirectly, without a need for separate helper AAV DNA.

3.1. Definitions

-   helper virus: a virus such as adenovirus, herpesvirus,    cytomegalovirus, Epstein-Barr virus, or vaccinia virus, which when    coinfected with AAV results in productive AAV infection of an    appropriate eukaryotic cell.-   helper AAV DNA: AAV DNA sequences used to provide AAV functions to a    recombinant AAV virus which lacks the functions needed for    replication and/or encapsulation of DNA into virus particles. Helper    AAV DNA cannot by itself generate infectious virions and may be    incorporated within a plasmid, bacteriophage or chromosomal DNA.-   helper-free virus stocks of recombinant AAV: stocks of recombinant    AAV virions which contain no measurable quantities of wild-type AAV    or undesirable recombinant AAV.-   rAAV vector: recombinant AAV which contains foreign DNA sequences    and can be produced as infectious virions.-   a lower case “p” placed in conjunction with the name of a virus,    e.g., sub201 to form psub201, denotes the virus inserted into a    plasmid.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Diagrams of AAV-containing plasmid DNAs and analysis of AAVrescue from transfected plasmid DNAs. (A) Structure of psub201, pAAV/Adand pAAV/neo. pEMBL8(+) sequences are shaded, AAV inverted terminalrepeats (ITR) are open and adenovirus (Ad) ITRs are solid. XbaI cleavagesites separating ITR sequences from AAV coding or neomycin resistance(SV-neo) sequences are indicated. (B and C) Blot analysis of replicatingDNA that contains AAV-specific or SV-neo sequences. Low-molecular-weightDNA was prepared at 40 hr after HeLa cells were transfected withindicated plasmid DNAs, or infected with sub201 plus AAV/neo virus, andsimultaneously infected with adenovirus at a multiplicity of 10pfu/cell. After electrophoresis, DNA fragments were transferred tonitrocellulose and probed with either the XbaI fragment from psub201containing only AAV-specific coding sequences (AAV probe, panel B) orthe XbaI fragment from pAAV/neo containing only SV-neo coding sequences(neo probe, panel C). AAV-specific monomer (m) and dimer (d) DNAs arelabeled.

FIG. 2. Analysis of DNA sequences present in recombinant stocks ofAAV/neo virus. HeLa cells were transfected with pAAV/neo plus pAAV/AdDNA as helper in the presence of adenovirus. 40 hr later, lysates wereprepared, adenovirus was inactivated by heating at 56° C. for 30 min,and AAV virions were subjected to equilibrium density centrifugation.Gradients were fractionated, DNA was extracted from each fraction, andsubjected to DNA blot analysis using, first, an AAV-specific codingsequence as probe DNA (top panel), and, then after stripping thenitrocellulose filter, a neo-specific sequence as probe (bottom panel)The two probe DNAs were labeled to nearly identical specific activities.The top of each gradient is at the right, and the right hand most lanecontains marker sequences homologous to the probe DNA (considerably moreneo than AAV marker was added) to provide positive controls.

FIG. 3. Electrophoretic analysis of AAV capsid proteins synthesized at40 hr after transfection of HeLa cells with pAAV/no-tr, pAAV/Ad orpsub201. Cells were simultaneously transfected with indicated plasmidDNAs and infected with adenovirus at a multiplicity of 10 pfu/cell. At40 hr after transfection/infection cells were labeled for 1 hr with[³⁵S]methionine, extracts were prepared, subjected toimmunoprecipitation with a monoclonal antibody prepared against AAVcapsids, and analyzed by electrophoresis in an SDS-containingpolyacrylamide gel. Bands corresponding to the three capsid proteinsVP1, VP2 and VP3 are indicated.

FIG. 4. Analysis of AAV-specific DNAs rescued from G418-resistant celllines produced by infection with AAV/neo virus. G418-resistant celllines containing rescuable AAV/neo genomes were infected with eitheradenovirus alone (Ad) or adenovirus plus sub201 (Ad+AAV). Low molecularweight DNA was prepared at 40 hr after infection and analyzed by DNAblot analysis using as probe DNA either the XbaI fragment from psub201containing only AAV-specific coding sequences (AAV probe, panel A) orthe equivalent fragment from pAAV/neo containing only SV-neo sequences(neo probe, panel B). Monomer (m) and dimer (d) AAV DNA molecules arelabeled.

FIG. 5. DNA blot analysis of AAV/neo-specific sequences integrated inG418-resistant cell lines. (A) Diagram of BamHl (B) and EcoRI (E)cleavage sites within AAV/neo DNA, and the affect of head-to-taildimerization on predicted cleavage products. (B and C) Blot analysis ofDNA prepared from rescuable and non-rescuable cell lines. High molecularweight DNA was cut with indicated enzymes and subjected to blot analysisusing the XbaI fragment from pAAV/neo containing only SV-neo sequencesas probe DNA. The positions of monomer (m) and dimer (d) AAV/neo DNAsare indicated.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for producing substantiallyhelper-free stocks of recombinant AAV and is directed toward producing aviral expression vector system with improved efficiency, applicability,definition, and safety relative to viral vector systems currentlyutilized. The method of the invention utilizes a two component systemcomprised of functionally, but not structurally, related rAAV genomes,one of which contains cis-acting sequences needed for DNA replicationand packaging and a segment of foreign DNA (the vector), but lacks DNAsequences encoding trans-acting products necessary for viralreplication; and the other (the helper AAV DNA) which provides thosetrans-acting viral functions not encoded by the vector, but whichcannot, itself, be replicated or incorporated into virions at detectablelevels. Importantly, the vector and the helper DNA should besufficiently nonhomologous so as to preclude homologous recombinationevents which could generate wild-type AAV.

For purposes of clarity, and not by way of limitation, the detaileddescription of the invention will be divided into the followingsections.

i) The Vector/Helper DNA Relationship

ii) Recombinant AAV Vectors

iii) Helper AAV DNA

iv) Production of Helper-Free rAAV Stocks

v) Utility of Helper-Free rAAV Stocks

5.1. The Vector/Helper AAV Virus Relationship

According to the present invention, helper-free stocks of recombinantAAV are generated using functionally related recombinant DNA vector andhelper AAV DNA constructs.

The rAAV vector of the invention comprises a recombinant AAV genome (thevector) which contains a foreign DNA sequence and is used to cotransfecteukaryotic cells together with helper AAV DNA in the presence of helpervirus. According to the present invention, only the rAAV vector issuccessfully replicated and packaged into infectious virions by saidtransfected eukaryotic cells, resulting in helper-free AAV vectorstocks.

The helper AAV DNA of the invention comprises a recombinant AAV genomewhich complements the functions necessary for replication and packagingof the rAAV vector, but shares no AAV sequences with the vector. Thehelper AAV DNA should be unable to replicate and/or unable to bepackaged into virions to a measurably significant degree. The inabilityof the helper AAV DNA to either recombine with rAAV vector sequences oritself form infectious virions is the crux of the present invention.

In order to facilitate construction of rAAV vector and helper AAV DNAsequences, a recombinant AAV molecule may be designed so as to provideconvenient cleavage sites which may be used to create “cassettes” of AAVsequence which may, when used in different combinations, be used togenerate vector or helper DNA constructs. For example, in a specificembodiment of the invention, psub201 may be used to derive both rAAVvector and helper recombinant molecules (as described more fully inSamulski et al., 1987, J. Virol. 61:3096-3101, which is incorporated inits entirety by reference herein). psub201 comprises an infectious AAVgenome inserted into plasmid PEMBL8, and contains engineered XbaIcleavage sites that flank the viral coding domain which do not affectreplication of the virus and which allow nonviral sequences to beinserted between the cis-acting viral terminal repeats; the entire viralgenome is flanked by PvuII cleavage sites that allow the entireinfectious viral chromosome to be excised from plasmid sequences invitro. According to the invention, a foreign DNA sequence may beinserted between the XbaI sites, leaving intact the AAV termini presentin the two flanking PvuII-XbaI fragments. The resulting rAAV vector iscapable of integrating into a host genome; complementary viral functionsmay be supplied by a helper DNA formed by inserting non-AAV sequences inthe PvuII-XbaI flanking terminal regions, leaving the coding regionbetween the two XbaI sites intact. The resulting helper DNA shares nocommon DNA sequences with the corresponding rAAV vector therebyprecluding the generation of wild-type virus by homologousrecombination. In a specific embodiment of the invention, as exemplifiedby Section 6, infra, adenovirus terminal sequences may be inserted intothe Pvu II-XbaI site in the place of the AAV terminal repeats. Theresulting helper DNA was itself found to be unable to replicate, butprovided sufficient AAV functions to allow high levels of replication ofrAAV vector molecules and production of helper free-stocks of rAAVvirions. According to this disclosure, other modified AAV molecules usedto derive rAAV vector or helper AAV DNA, bearing different combinationsof restriction enzyme sites at different, or similar regions of the AAVgenome, may be constructed by one skilled in the art.

5.2. Recombinant Adeno-Associated Virus Vectors

The recombinant AAV (rAAV) vectors of the present invention include anyrecombinant DNA molecule which incorporates sufficient regions of thewild-type AAV genome to permit replication of DNA, normal integrationinto, as well as excision from, the host cell genome, and should includea cis-acting packaging element. The rAAV vector itself need not containAAV genes encoding proteins, including those associated with DNA or RNAsynthesis or processing or any step of viral replication includingcapsid formation. In preferred embodiments of the invention, the rAAVvector retains only terminal AAV sequences necessary for integration,excision, replication, and packaging; comprising less than about 195base pairs of the AAV terminus. In a specific embodiment of theinvention, recombinant viruses were generated that contained only 191nucleotides of the AAV chromosome, and were derived from plasmid psub201DNA, as described in Samulski et al. (1987, J. Virol. 61: 3096-3101)which is incorporated by reference in its entirety herein.

According to the invention, the rAAV vector may be propagated inmicroorganisms, for example, as part of a bacterial plasmid orbacteriophage, in order to obtain large quantities of rAAV DNA which maybe utilized according to recombinant DNA methodology to generate novelconstructions. In a specific embodiment of the invention, the vector ispEMBL8(+).

The rAAV vector of the present invention may incorporate any foreign DNAsequence, including genes or portions of genes. It may be desirable toincorporate a gene with a readily detectable product (known in the artas a marker, recorder, or reporter gene) as part of the rAAV vectoralthough the invention is not limited to such constructs. Readilydetectable reporter genes may produce either tumorigenic ornon-tumorigenic products. Tumorigenic reporter genes could be utilized,but might present a significant health risk due to their oncogenicity.Non-tumorigenic reporter genes would include, but are not limited to,β-galactosidase, neomycin phosphoro-transferase, chloramphenicolacetyltranferase, thymidine kinase, luciferase, β-glucuronidase, andxanthine-guanine phosphoribosyl transferase, to name but a few.

According to the invention, if the foreign DNA of the rAAV vector is tobe expressed in host cells, a transcriptional control element, alsocalled a promoter/enhancer sequence, should be provided. Thepromoter/enhancer sequence may be widely active or may, alternatively,be tissue specific. The promoter/enhancer sequence may be derived from anon-AAV source or may be an AAV promoter provided that no sequences areshared by helper AAV DNA. Promoter/enhancer sequences which might beused to control foreign gene expression, provided for in the presentinvention, include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, 1981, Nature 290:304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., 1980, Cell 22:787-797), the herpesvirus thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:144-1445), the regulatory sequences of the metallothionine gene(Brinster et al., 1982, Nature 296:39-42); and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science235:53-58); alpha 1-antitrypsin gene control region which is active inthe liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315:338-340, Kollias et al., 1986, Cell 46:89-94; myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-712); myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-1378).

The desirable size of inserted non-AAV or foreign DNA is limited to thatwhich permits packaging of the rAAV vector into virions, and depends onthe size of retained AAV sequences. AAV-2 is 4675 base pairs in length(Srivastrava et al., 1983, 5. Virol. 45:55-564). Tratschen et al. (1984,Mol. Cell. Biol. 4:2072-2081) constructed an AVBcCAT AAV genome that was3 percent (approximately 150 nucleotides) longer than the wild-type AAVgenome, and found that AVBcCAT could be packaged into virions. An AAVgenome too large to be packaged resulted from insertion of a 1.1kilobase pair fragment of bacteriophage λ inserted into a nonessentialregion of AAV (Samulski and Shenk, personal communication, cited inHermonat and Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466:6470).Thus, the total size of the rAAV to be packaged into virions should beabout 4800-5000 nucleotides in length.

It may be desirable to exclude portions of the AAV genome in the rAAVvector in order to maximize expression of the inserted foreign gene. Forexample, it has been observed (Hermonat and Muzyczka, 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6466-6470; Lebkowski et al., 198, Mol. Cell. Biol.8:3988-3996; Tratschin et al., 1985, Mol. Cell. Biol. 5:3251-3260) thatthe number of drug-resistant colonies produced by transduction generallydid not increase linearly as the multiplicity of AAV recombinant viruswas increased. This may have resulted, at least in part, from theinhibitory effects of AAV-coded rep functions. In contrast, using thehelper-free rAAV stock of the present invention, wherein the rAAV vectorcarried the gene for neomycin resistance (see section 6, infra, andTable II) the number of drug-resistant transductants responded almostlinearly to the input multiplicity of recombinant virus. Therefore, itmay be desirable to exclude rep encoding sequences from the rAAV vector,as was done in the specific embodiment of the invention describedherein.

5.3. Helper Adeno-Associated DNA

The helper AAV DNA of the present invention i) provides viral functionsnecessary for the replication of rAAV vector and its packaging intoinfectious virions ii) shares no AAV sequences with rAAV vector, andiii) is not itself replicated or assembled into viral particles to ameasurable degree. The helper AAV DNA may contain the entire codingregion of the AAV genome or a portion thereof, provided that the aboverequirements, i-iii), are met.

According to the invention, helper AAV DNA may be propagated inmicroorganisms, for example, as part of a bacterial plasmid orbacteriophage. Portions of the AAV genome may be incorporated into arecombinant plasmid, bacteriophage, etc. by methods well known in theart. In a specific embodiment of the invention, the vector is pEMBL8(+).

In order to provide viral functions necessary for the replication ofrAAV vector and its packaging into infectious virions, the helper AAVDNA must contain cis-acting elements which promote the transcription ofdesirable viral gene products; such elements may be AAV or non-AAVderived and include but are not limited to the promoter sequences listedin section 5.2, supra. Sequences which facilitate the translation of AAVproteins may also be included.

Any sequences may be incorporated into the helper AAV virus that may beutilized to increase production of rAAV vector virions. Accordingly,transcriptional enhancer or repressor elements may be incorporated intothe helper DNA, including, but not limited to, such elements derivedfrom AAV or adenovirus.

The helper AAV DNA cannot itself be incorporated into infectious virionsto any significantly measurable degree. According to the invention,helper DNA can be excluded from virion formation by virtue of i) failureto replicate to form a discrete recombinant AAV genome and ii) failureto be packaged into infectious virions. Methods of preventing packagingof rAAV helper DNA include constructing a rAAV helper DNA genomeprohibitively large, for example, greater than approximately 5.8kilobase pairs in length or constructing an rAAV helper DNA which lacksa cis-acting packaging sequence.

In a specific embodiment of the invention, a rAAV helper DNA may beconstructed by removing the AAV terminal sequences of psub20l (Samulskiet al., 19-87, J. Virol. 61:3096-3101, incorporated by reference herein)from the Xba I-Pvu II sites and by inserting, into each of the Xba Isites, an Eco RI-Xba I fragment consisting of the 107 terminal basepairs of adenovirus type 5 DNA using methods known in the art.Unexpectedly, the introduction of the adenovirus sequences has beenobserved to result in a high efficiency of replication of rAAV vector incotransfected cells (see Section 6, infra).

In an additional embodiment of the invention, the AAV helper DNA may beincorporated into a cell line, thus bypassing the need to cotransfectAAV helper DNA and rAAV vector sequences. Instead, transfection of theAAV helper DNA-containing cell line with rAAV vector DNA plus infectionwith the helper adenovirus would directly result in production ofhelper-free rAAV virus. The present invention provides for such AAVhelper DNA-containing cell lines, which may be created by transfecting acell line permissive for AAV growth with AAV helper virus DNA, and thenidentifying cells which have stably integrated the transfected DNAsequences, either by identifying cells which express AAV proteins or anyother gene, i.e. a reporter gene incorporated into the AAV helper virusDNA or cotransfected with it. The presence of AAV helper virus sequencesmay be corroborated by propagating selected cells, and then testing forAAV helper virus sequences by hybridization techniques, i.e. Southernblot, “dot blot”, etc. It may be desirable to control the expression ofAAV gene products if their expression proves to be toxic to thetransfected cells. For example, and not by way of limitation, theexpression of AAV products from the helper AAV virus DNA may be putunder the control of an inducible promoter/enhancer. For example, themouse mammary tumor virus promoter/enhancer, which may be induced bytreatment with glucocorticoids, may be used to control expression of AAVproducts; cells carrying this construct would normally express lowlevels of AAV products, but could be induced to higher levels ofexpression with dexamethasone prior or simultaneous to introduction ofrAAV DNA.

5.4. Production of Helper-Free Recombinant AAV Stocks

The phrase “helper-free recombinant AAV stocks”, according to theinvention, is construed to mean stocks of infectious virions whichcontain, virtually, only rAAV vector (as defined supra and in section3.1) and contain no significant quantities of wild-type AAV orundesirable recombinant AAV (presumably derived from rAAV helper DNAsequences).

According to the method of the invention, helper-free recombinant AAVmay be produced by cotransfecting an appropriate cell type with rAAVvector and helper AAV DNA, in the presence of a helper virus, such asadenovirus or herpes virus.

Cotransfection may be performed by the DEAE dextran method (McCutchenand Pagano, 1968, J. Natl. Cancer Inst. 41:351-357), the calciumphosphate procedure (Graham et al., 1973, J. Virol. 33:739-748) or byany other method known in the art, including but not limited tomicroinjection, lipofection, and electroporation. Cotransfection may beaccomplished using helper virus infected cells, or may be performedsimultaneously with, or prior to, viral infection. If adenovirus is usedas helper virus, a desirable multiplicity of infection may be betweenabout 5 and 10. Amounts of rAAV vector and helper DNA used intransfection are approximately 0.2 to 10 μg of DNA per 10⁶ cells, butmay vary among different DNA constructs and cell types. Cells suitablefor transfection of recombinant AAV include any cell line permissive forAAV infection, including, but not limited to HeLa cells or human 293cells (human embryonic kidney cells transformed with a fragment ofadenovirus 5 DNA).

Several days following transfection, a rAAV virus stock may be generatedby i) producing a cell lysate, ii) inactivating helper virus (forexample, heat inactivation of adenovirus at about 56° C. forapproximately 30-45 minutes), and iii) purifying rAAV virions from otherelements of the cell lysate according to methods known in the art, suchas ultracentrifugation using, for example, a cesium chloride gradient.

Corroboration of the helper-free nature of the resulting rAAV stock maybe accomplished by the isolation of low-molecular weight DNA accordingto the method of Hirt (1967, J. Mol. Biol. 26:365-369), with subsequentevaluation for the presence of appropriate DNA sequences, using methodswell known in the art.

5.5. Utility of Helper-Free Recombinant AAV Stocks

The helper-free recombinant AAV stocks produced according to the methodof the invention may then be used to infect any cell permissive for AAVinfection, including primary cells, established cell lines, a tissue, oran organism. It has been shown that introduction of rAAV by infection(using virions) is about two orders of magnitude more efficient thantransfection with rAAV DNA (Tratshen et al., 1985, Mol. Cell. Bio.5:3251-3260). The method of the invention thereby provides a moreefficient means of introducing a rAAV which is virtually free ofcontaminating AAV into host cells. Prior to the invention, viral stockswere found to be contaminated, to various degrees, with helpersequences, and therefore the purity of rAAV construct being introducedinto the cells by infection could not be assured.

In order to identify cells which, consequent to rAAV infection, expressforeign DNA sequences, it may be desirable to incorporate a gene whichencodes a selectable or detectable product, for example, the neomycinresistance gene (Southern and Berg, 1982, J. Mol. Appl. Genetics1:327-341) or the hygromycin resistance gene (Blothlinger andDiggelmann, 1984, Mol. Cell. Biol. 4:2929-2931).

The capability to produce helper-free rAAV stocks enables the executionof techniques which were inefficient or impossible in the presence ofcontaminating AAV sequences. Since wild-type AAV is not present toinhibit high efficiency transformation of cells infected with the rAAVvirion stock, nearly all of the cells exposed to rAAV can be infected(Table III). The rAAV DNA can then be integrated into the chromosomes ofrecipient cells. This high efficiency gene transfer may be especiallyuseful if one wishes to introduce a foreign DNA sequence into a rarecell type within a mixed cell population. Rare stem cells within thetotal population of hematopoietic cells obtained from the bone marrow ofan animal or a human are an example of such a rare cell type. Since therAAV virion stock can successfully infect nearly all cells within apopulation, rare stem cells may be successfully infected.

In a specific embodiment of the invention, it was, unexpectedly, foundthat the replacement of the AAV terminal repeated sequences withadenovirus 5 DNA sequences resulted in a high level of replication ofrAAV vector in cotransfected cells.

According to a specific embodiment of the invention, a rAAV vector whichlacks all rAAV coding sequences (and therefore maximizes the potentialsize of foreign DNA transduced) can be used to produce pure viral stocksand, surprisingly, despite its minimal amount of AAV sequence, cansuccessfully integrate in rescuable form into cellular chromosomes.

In addition, the ability to produce helper-free stocks of rAAV enablesmore controlled rescue of incorporated AAV provirus, requiring additionof both wild-type AAV and adenovirus. Prior to the method of theinvention, because of contaminating helper AAV virus, rescue of proviruscould proceed in a less controlled manner, requiring only helperadenovirus.

Finally, the method of the invention significantly augments the utilityof adeno-associated virus as a transducing vector. The broad host range,capacity to readily insert and/or be excised from cellular DNA, abilityto act as part of a “shuttle vector” between prokaryotic and eukaryoticcells, and effective absence of pathogenicity are advantages of the AAVtransducing system hitherto offset by the inability to generatehelper-free virus. The method of the invention overcomes the obstaclepresented by contaminated viral stocks, and allows for expansion of therAAV transducing vector system. In the future, rAAV may prove to be thevector of choice for gene transfer into organisms.

6. EXAMPLE Helper-Free Stocks of Recombinant Adeno-Associated Viruses(AAV): Normal Integration does not Require Viral Gene Expression

Recombinant AAV virus stocks invariably have been contaminated withhelper AAV virus used to provide acting functions required for AAVreplication and encapsidation. In this report we describe a method forproduction of recombinant AAV virus stocks that contain no detectablewild-type helper AAV virus. The recombinant viruses contain only theterminal 191 nucleotides of the viral chromosome, demonstrating that allcis-acting AAV functions required for replication and virion productionare located within that region. Helper-free virus stocks were able tostably introduce a drug-resistance marker gene into a high percentage(70%) of infected cells. Recombinant viral DNAs carrying a drugresistance marker gene were integrated into the cellular genome and thenexcised and replicated when the cells were subsequently infected withwild-type AAV plus adenovirus. Thus, the AAV termini are sufficient fornormal integration of the AAV chromosome into a host cell genome. No AAVgene expression is required to establish a latent infection.

6.1. Materials and Methods 6.1.1. Plasmids and Viruses

psub201 (Samulski et al., 1987, J. Virol. 61:3096-3101), an infectiousclone of AAV type 2 DNA, served as the parent to all plasmid constructsdescribed in this report. pAAV/neo and pAAV/hyg were prepared bysubstituting the AAV coding region in psub201 with either theneomycin-resistance gene (Southern and Berg, 1982, J. Mol. Appl.Genetics 1:327-341) or hygromycin resistance gene (Blochlinger andDiggelman, 1984, Mol. Cell. Biol. 4:2929-2931) controlled by the SV40early promoter. The drug-resistance genes were substituted between thetwo XbaI sites present in psub201 to generate recombinant viral DNAs inwhich 191 bp segments from the termini of sub201 bracket either theneomycin- or hygromycin-resistance gene. pAAV/no-tr was generated bydeleting the 191 base pair AAV termini from psub201 DNA using XbaI andPvuII cleavage sites which bracket the terminal segments. pAAV/Ad wasproduced by inserting into each of the two XbaI sites of pAAV/no-tr anEcoRI-XbaI fragment consisting of the 107 terminal base pairs ofadenovirus type A DNA which were derived from a cloned, left-terminalfragment of the viral chromosome. sub201 is a phenotypically wild-typederivative of AAV type 2 (Samulski et al., 1987, J. Virol.61:3096-3101). AAV/neo and AAV/hyg virus stocks were generated bycotransfection of adenovirus-infected cells with pAAV/neo or PAAV/hygplus pAAV/Ad. The concentration of sub201, AAV/neo or AAV/hyg virusparticles in virus stocks was determined by extracting and quantifyingDNA from a known volume of virus stock. A virus lysate was heat-treatedat 56° C. for 30 min to inactivate the adenovirus, treated with DNaseI(10 μg/ml) at 37° C. for 15 min to degrade nonvirion DNA, heated at 68°C. for 10 min to inactivate DNase I, treated with proteinase K (100μg/ml) in SDS (0.1%) at 37° C. for 3 hrs, and then nucleic acids wereextracted with phenol/chloroform and precipitated with ethanol. DNA wasresuspended in low salt buffer, denatured by boiling, diluted into 10volumes of ice cold 10×SSC (1×SSC is 0.15M NaCl, 0.015M sodium citrate),applied to a nitrocellulose sheet using a dot blot apparatus and probedusing a [³²P]labeled DNA common to all three viruses (AAV terminalsequence). The intensities of dots were compared to those of standardcurve generated by assaying dilutions of an AAV DNA preparation of knownconcentration.

6.1.2. Cell Cultures

HeLa cells were from the American Type Culture Collection (Rockville,Md.). Detroit 6 cells are a human lung carcinoma cell line (Berman etal., 1955, Blood 10:896-911) that do not contain integrated AAVsequences (Berns et al., 1975, Virology 68:556-560). Both cell lineswere propagated in medium containing 10% calf serum.

6.1.3. DNA Replication and Excision Assays

To assay AAV-specific DNA replication, HeLa cells were transfected withcircular plasmid DNAs by the DEAE-dextran procedure (McCutchan et al.,1968, J. Natl. Cancer Inst. 41:351-357). 1.0 μg of each DNA (recombinantand helper) was included in the transfection mixtures as well asadenovirus type 5 virions at a multiplicity of 10 pfu/cell. Excision andrescue was assayed by infection of AAV-containing Detroit 6 cells witheither adenovirus alone or with both adenovirus plus wild-type AAV(sub201). For both replication and excision/rescue assay, lowmolecular-weight DNA was isolated by the procedure of Hirt (1967, J.Mol. Biol. 26:365-369 at 40 hr after transfection/infection, andanalyzed by DNA blot analysis for the presence of AAV-specific DNAs.

6.1.4. DNA Blot Analysis

High molecular weight DNA was prepared, 10 μg portions were digestedwith the appropriate restriction enzymes, digests were fractionated byelectrophoresis on a 1% agarose gel, transferred to nitrocellulose, andhybridized using [³²P] labeled probe DNAs prepared by nick-translation(Maniatis et al., 1982 in “Molecular Cloning, a laboratory manual”, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

6.1.5. Protein Analysis

For analysis of AAV capsid proteins, HeLa cells were simultaneouslytransfected with AAV-specific plasmid DNAs by the calcium phosphateprocedure (Graham et al., 1973, Virology 52:456-467) and infected withadenovirus. Cultures were labeled for 1 hr with [³⁵S]methionine (100μCi/ml; 1,100 Ci/mmole) 40 hr later and then subjected toimmunoprecipitation with a monoclonal antibody prepared against purifiedAAV capsid proteins (L.-S. Chang, S. Pan. R. J. Samulski and T. Shenk,in preparation). Immunoprecipitation and electrophoresis were aspreviously described (Sarnow et al., 1982, Cell 28:387-394).

6.2. Results 6.2.1. Recombinant AAV Stocks that Contain No DetectableHelper AAV

We wished to determine whether AAV gene expression is required fornormal integration of AAV DNA into the genome of a host cell. To askthis question, two conditions had to be met. First, it was necessary toconstruct a recombinant virus that contained cis-acting but notrans-acting AAV functions. Second, it was necessary to generate a stockof the recombinant virus containing no helper AAV particles that couldprovide trans-acting functions during a subsequent infectious cycle.

Cis-acting AAV sequences appear to reside entirely in and near theterminal repeated sequences at the ends of the viral chromosome(McLaughlin et al., 1988, J. Virol. 62:1963-1973). Therefore, tworecombinant viruses were produced that contained AAV termini bracketingmarker genes.

psub201 DNA (Samulski et al., 1987, J. Virol. 61:3096-3101 was used as arecipient for non-viral sequences. This is an infectious clone of AAVwhose virus-specific insert (FIG. 1A) differs from wild-type AAV in thattwo XbaI cleavage sites have been added at sequence positions 190 and4484, and the right-end 191 base pairs of the viral DNA (the XbaI siteat position 4484 to the right terminus) have been substituted for theleft-end 190 base-pair domain (left terminus to the XbaI site atposition 190). Since the viral DNA includes terminal repeats of 145 basepairs, the modification results in the substitution of 46 base pairsunique to the right-end domain of the AAV chromosome (sequence position4485 to 4530) for 45 base pairs present near the left end (sequenceposition 146 to 190). When HeLa cells were transfected with psub201 DNAin the presence of adenovirus, the AAV chromosome was excised fromplasmid sequences and replicated normally (Samulski et al., 1987, J.Virol. 61:3096-3101).

The pair of XbaI cleavage sites in psub201 provided the opportunity tosubstitute the entire AAV coding region with non-viral sequences whichwere then bracketed by the AAV terminal repeats. Either theneomycin-resistance or hygromycin-resistance coding regions regulated bythe SV40 early transcriptional control region was substituted for theAAV coding region to produce pAAV/neo (FIG. 1A) and pAAV/hyg,respectively. These recombinant constructs contained the known AAVcis-acting but no trans-acting functions.

Two helper DNAs were produced. The first was terminal pAAV/no-tr (noterminal repeat) since it contained only the AAV coding sequencesinternal to the XbaI cleavage sites of psub201. The second was termedpAAV/Ad since it contained the adenovirus type 5 terminal sequence (107bp) in place of the normal AAV termini (FIG. 1A). Neither helper DNAcould be packaged into AAV virions since they lacked the terminalcis-acting domain required for this function, and neither constructcontained sequences present in pAAV/neo or pAAV/hyg, precludinghomologous recombination between helper and recombinant viral DNAs.

pAAV/no-tr and pAAV/Ad were compared to wild-type AAV DNA (psub201) fortheir ability to provide helper function to pAAV/hyg (Table 1).pAAV/hyg, as circular plasmid DNA, was transfected into HeLa cellstogether with various helper plasmids in the presence of adenovirus(d1309, 10 pfu/cell). When maximal cytopathic effect occurred (72 hr),cultures were harvested, freeze-thawed three times, and heat treated(56° C., 45 min) to inactivate adenovirus. Dilutions of the lysates werethen used to infect subconfluent HeLa cells, and hygromycin-resistant(Hyg^(R)) colonies were selected. TABLE I AAV/hyg Yields Produced UsingVarious Helper Plasmids to Provide Trans-acting AAV Functions. HelperPlasmid Hyg^(R) Colonies/ml Lysate pAAV/no-tr 3 × 10² pAAV/Ad 3 × 10⁴psub201 5 × 10⁵By this assay, psub201 DNA was the most efficient helper. pAAV/no-tr wasvery inefficient, while pAAV/Ad performed at an intermediate level.Contransfection with pAAV/Ad DNA generated a yield of AAV/hyg virusreduced by a factor of 10-20 as compared to stocks generated bycotransfection with psub201 DNA. It produced 3×10⁴ hygromycin-resistantcolonies per ml lysate (Table I).

Accumulation of AAV-specific DNAs was monitored in HeLa cells infectedwith adenovirus and simultaneously cotransfected with pAAV/neo DNA andeither psub201. or pAAV/Ad helper DNA (FIG. 1). Low-molecular-weightDNAs were extracted at 40 hr after infection/transfection and subjectedto DNA blot analysis. The blots were first probed using a [³²P]labeledDNA comprising AAV coding sequences which detected helper virus DNAs butnot AAV/neo DNA (FIG. 1B). Analysis with this probe indicated that AAVhelper DNA was excised and amplified in cells transfected with psub201DNA but not pAAV/Ad DNA. The adenovirus termini could not substitute forthe cis-acting functions present in AAV termini. Next, the blots wereprobed using a [³²P]labeled DNA comprising coding sequences from theneomycin-resistance gene which detected AAV/neo DNA but not helper virusDNAs (FIG. 1C). The recombinant viral DNA was excised from pAAV/neo DNAand amplified to a similar extent in the presence of either helpervirus.

The AAV/neo stocks obtained using either pAAV/Ad or psub201 DNA ashelper were then used to infect HeLa cells in the presence ofadenovirus. The recombinant virus stock prepared using psub201 DNA ashelper produced replicating AAV/neo DNA (FIGS. 1B and C, P2), since thestock also contained sub201 virus to provide AAV helper functions. Incontrast, the recombinant virus stock prepared using pAAV/Ad DNA ashelper did not generate replicating AAV/neo DNA (data not shown). Thismakes sense since this stock was predicted to contain no helper virus toprovide trans-acting AAV functions. To further confirm that no helpervirus was present in stocks of AAV/neo prepared using pAAV/Ad as helperDNA, a stock of the recombinant virus was subjected to equilibriumdensity centrifugation, fractions were collected, and probed for thepresence of neo and AAV coding sequences (FIG. 2). Only neo codingsequences were detected in the portion of the gradient (fractions 9, 10,and 11) containing virions.

pAAV/Ad was originally constructed to test the possibility that theadenovirus termini might mediate replication of the AAV sequences in thepresence of the adenovirus helper virus. If the adenovirus terminimediated any amplification of AAV/Ad sequences, it was with poorefficiency since AAV/Ad DNA was not detected in the DNA blot analysisdescribed above (FIG. 1B). Nevertheless, pAAV/Ad served as asignificantly better helper than pAAV/no-tr (Table I).

Even though AAV/Ad DNA was not detectably amplified, AAV gene productswere expressed at higher levels in cells transfected with pAAV/Ad thanthose receiving pAAV/no-tr. This was demonstrated by immunoprecipitationof AAV capsid proteins from cells labeled with [³⁵S]methionine for 1 hrbeginning at 40 hr after transfection (FIG. 2). Cells infected withadenovirus and transfected with pAAV/Ad DNA produced nearly as much AAVcapsid protein as cells that received the infectious AAV clone, psub201.This probably results from enhanced transcription of the AAV genome inthe presence of transcription-promoting elements within the adenovirusterminal repeats (Hearing and Shenk, 1983, Cell 33:695-703).

6.2.2. Normal Integration of Recombinant DNAs in the Absence of AAV GeneExpression

Using pAAV/Ad DNA as a helper, it was possible to generate stocks ofrecombinant viruses that contained no helper virus. The amount ofrecombinant virus in these stocks was determined by quantitation ofhybrid DNA extracted from virions (see Section 6.1, Materials andMethods). It was also possible to quantitate the recombinant virus usinga biological assay (Table 2). pAAV/neo was transfected into HeLa cellstogether with pAAV/Ad helper plasmid in the presence of adenovirus(d1309, 10 pfu/cell). When maximal cytopathic effect occurred, cultureswere harvested, freeze-thawed three times and then heat treated (56° C.,45 min) to inactivate adenovirus. The concentration of AAV/neo particlesin the resulting stock was then determined by dot blot analysis usingdilutions of known AAV DNA concentrations to establish a standard curve.AAV/neo was then used to infect subconfluent HeLa cells in the presenceor absence of sub201, and G418-resistant (G418^(R)) colonies wereselected. Each value presented in the table is the average of 10independent transduction mixtures of 10⁴ cells each. The number ofcolonies increased in proportion to the input multiplicity of AAV/neovirions (Table 2, experiment 1). TABLE II AAV/neo Transformation of HeLaCells in the Absence and Presence of Helper Virus to ProvideTrans-Acting AAV Functions AAV/neo sub201 G418^(R) Colonies/10⁴(Particles) (Particles) cells Experiment 1. 10³ — 2 10⁴ — 18 10⁵ — 85Experiment 2. 10⁴ — 51 10⁴ 10³ 92 10⁴ 10⁴ 220 10⁴ 10⁵ 87The number of G418-resistant colonies could be increased further whenthe input multiplicity of AAV/neo virus was held constant and increasingamounts of wild-type AAV (sub201) were added to the infecting mixture(Table II, experiment 2). However, the maximal enhancement was only4-fold, and the total number of drug-resistant colonies decreased at thehighest input levels of sub201. Thus, the efficiency of transduction byAAV/neo virions was not greatly enhanced by the presence of trans-actingAAV functions.

The highest multiplicity of AAV/neo virus used in the previousexperiments was 10 particles/cell. About 20 to 100 wild-type AAVparticles constitute a single infectious unit (McLaughlin et al., 1988,J. Virol. 62:1963-1973; Tratschin et al., 1985, Mol. Cell. Biol.5:3251-3260). Thus it seemed possible that the efficiency oftransduction could be enhanced by increasing the input multiplicity ofrecombinant virions in the absence of helper virus. Accordingly, AAV/neovirus particles from 30 plates (10 cm diameter) of 293 cells transfectedwith pAAV/neo plus pAAV/Ad and infected with adenovirus were purifiedand concentrated by equilibrium density centrifugation, the gradient wasfractionated, aliquots were dialyzed into tris-buffered saline, and theconcentration of AAV/neo particles in peak fractions was determined bydot blot analysis as described in the legend to Table II. A total ofapproximately 1×10⁸ AAV/neo particles was obtained. The concentratedstock was then used to infect 10⁴ Detroit 6 cells at a multiplicity of1000 particles/cell. 24 hr later, cells were removed from the plate,counted and plated at the cell densities indicated. After an additional24 hr. G418 was added. Drug-resistant colonies were counted two weekslater. Approximately 70% of the infected cells gave rise toG418-resistant colonies (Table III). TABLE III Transduction of Detroit 6Cells with AAV/neo Virions at a High Multiplicity of Infection in theAbsence of Helper Virus. Number of Number of Infected CellsG418-Resistant Colonies 9 × 10³ >6000 1 × 10³ 650 1 × 10² 72

Thus, the efficiency of transduction can be dramatically increased byinfecting with recombinant virus at high multiplicity in the absence ofhelper virus.

Next, the physical structure of the AAV/neo chromosome was examined inG418-resistant cell lines. 17 drug-resistant clones were prepared fromDetroit 6 cells infected with helper-free AAV/neo virus. No AAV DNA wasdetected by DNA blot analysis of low-molecular-weight DNA prepared fromthe cell lines by the procedure of Hirt, 1967, J. Mol. Biol. 26:365-369(data not shown). AAV sequences were present in very large DNA fragmentswhen high molecular weight cellular DNA was subjected to DNA blotanalysis (data not shown). Further, detailed DNA blot analysis of threenon-rescuable and one rescuable cell line (FIG. 5B, discussed below),revealed fragments of appropriate size to represent viral/host DNAjunctions. Therefore, the AAV/neo DNA present in each of the cells lineswas almost certainly integrated into the host cell chromosome.

Normally AAV DNA can be induced to excise from the host cell chromosomeand replicate by infection with adenovirus. As expected, AAV/neo DNA wasnot rescued from any of the 17 lines by infection with adenovirus alone(data for 7 lines is displayed in FIG. 4). AAV/neo DNA was rescued from7/17 lines by coinfection with both wild-type AAV (sub201) andadenovirus (FIG. 4). This experiment supported the earlier conclusionthat no wild-type AAV was present in the AAV/neo virus stock since nonewas present in any of the drug-resistant cell lines. Further, itdemonstrated that AAV/neo DNA could be “properly” integrated in theabsence of AAV gene expression in the sense that it could be excisedwhen AAV plus adenovirus-coded products were subsequently provided.

AAV DNA which has integrated into the cellular chromosome in a rescuablestate often exists as head-to-cell concatemers (Cheung et al., 1980, J.Virol. 33:739-748; Laughlin et al., 1986, J. Virol. 60:515-524;McLaughlin et al., 1988, J. Virol. 62:1963-1973). DNA blot analysis wasperformed to determine whether this was also the case for cell linesthat harbored rescuable AAV/neo DNA (FIG. 5). Head-to-tail concatemersof AAV/neo DNA can be identified by digestion of high molecular weightDNA with the EcoRI and BamHl endonucleases. Head-to-tail concatemerswill generate unit-length DNA when cleaved with only one of the twoenzymes and a shorter than unit-length fragment when cleaved with both(FIG. 5A). Inserts of single AAV/neo molecules or head-to-headconcatemers will not generate the predicted fragments. Threenon-rescuable cell lines contained no detectable head-to-tailconcatemers by this assay (FIG. 5B). All seven rescuable cell linesgenerated AAV/neo DNA fragments of the size predicted for head-to-tailconcatemers (FIGS. 5B and C). Thus, rescuable cell lines were generatedin the absence of trans-acting AAV function which contain tandemlyduplicated molecules of AAV/neo DNA arranged in a head-to-tailorientation.

6.3. Discussion 6.3.1. Helper Virus-Free Stocks of Recombinant Viruses

Earlier studies attempted to minimize the level of contaminating helperAAV viruses in recombinant AAV stocks either by using a helper DNA toolarge to be encapsidated (Hermonat and Muzyczka, 1984, Proc. Natl. Acad.Sci. U.S.A. 81:6466-6470; McLaughlin et al., 1988, J. Virol.62:1963-1973) or by employing a helper DNA lacking all or portions ofone or both terminal AAV sequences (Lebkowski et al., 1988, Mol. Cell.Biol. 8:3988-3996). However, homologous recombination events betweenhelper and hybrid DNA molecules generated wild-type AAV DNA that couldthen be amplified and encapsidated.

The experiments reported here demonstrate that it is possible togenerate stocks of recombinant AAV virions that do not containdetectable helper virus. This was achieved by employing a helper virusthat lacked all known cis-acting AAV replication and encapsidationfunctions (pAAV/Ad, FIG. 1A). Further, the helper AAV and recombinantAAV DNA pair contained no sequence in common, minimizing the opportunityfor recombination that could generate a packageable helper chromosome.The recombinant viruses contained only the right terminal 191 bp of theAAV chromosome bracketing both ends of a foreign DNA (AAD/eno andAAV/hyg, FIG. 1A), while the helper DNA lacked the same terminalsequence (pAAV/Ad, FIG. 1A).

A variety of observations support the conclusion that the stocks ofAAV/neo and AAV/hyg contained no detectable helper virus such as AAV/Ador a wild-type-like virus generated by non-homologous recombination.First, no replication of AAV/Ad DNA or any other molecule containing theAAV coding region was detected in HeLa cells cotransfected with pAAV/Adand pAAV/neo DNA in the presence of adenovirus (FIG. 1B). Second, theresulting AAV/neo virus stock could not be propagated through a secondround of infection by coinfection with adenovirus alone (data notshown), indicating it did not contain any helper or wild-type-like AAVparticles to provide trans-acting AAV functions. Third, no AAV codingsequences were detected in peak fractions of AAV/neo virions that weresubjected to equilibrium centrifugation in a cesium chloride gradientand then assayed by DNA blot analysis (FIG. 2). Fourth, none of theDetroit 6 cell lines produced by transduction of G418 resistance withthe AAV/neo stock contained either rescuable AAV DNA or AAV trans-actingfunctions (FIGS. 4A and 4B).

The 191 bp segments which included the entire 145 bp terminal repeats ofthe AAV genome contained the cis-acting signals needed to achieveexcision and replication of the recombinant DNAs at normal efficiencysubsequent to transfection. AAV/neo DNA accumulated to about the samelevel as sub201 DNA within transfected HeLa cells (FIGS. 1B and 1C). Itis not possible to determine from our present results whether therecombinant genomes were encapsidated at normal efficiency. However, itis clear, given our ability to produce AAV/neo and AAV/hyg virions, thatthe right end 191 bp of the AAV chromosome (this sequence is present atboth ends of the recombinant DNAs) contains sufficient cis-actinginformation to signal packaging at some level. These constructs furtherrefine the localization of the putative cis-acting AAV packaging elementfrom 284 bp (McLaughlin et al., 1988 supra) to 191 bp (AAV/neo, FIG.1A).

The AAV terminal sequences were originally substituted with adenovirusterminal sequences in pAAV/Ad since it seemed possible that theadenovirus DNA replication origins present in the terminal sequenceswould respond to adenovirus-coded replication functions and serve toamplify the helper chromosome. This did not occur at detectable levelseither when pAAV/Ad DNA was introduced as a circular DNA (FIG. 1B) orwhen AAV/Ad sequences were cleaved from the plasmid and used totransfect cells as linear molecules (data not shown). As luck would haveit, however, the adenovirus termini substantially enhanced expression ofAAV-specific proteins from pAAV/Ad DNA as compared to pAAV/no-tr thatcontained neither adenovirus nor AAV terminal sequences (FIG. 3). Thisenhanced expression is likely due to transcriptional enhancing elementsbelieved to preside in the adenovirus terminal repeats (Hearing andShenk, 1983, Cell 33:695-703).

6.3.2. Normal Integration without AAV Gene Expression

Earlier studies of drug-resistant cell lines generated by transductionwith a stock of recombinant AAV (e.g., AAV carrying aneomycin-resistance gene) containing wild-type helper virus haveidentified lines that contain a recombinant but no wild-type AAVchromosomes (e.g., McLaughlin et al., 1988, J. Virol. 62:1963-1973).However, it is not possible to be certain from this result whethertrans-acting AAV functions were expressed and mediated the integrationof the AAV recombinant. Perhaps the original cell received bothrecombinant and wild-type viruses, although the wild-type genome wasexpressed, only the recombinant chromosome became integrated.

The experiments reported here clearly demonstrate that a recombinantvirus (AAV/neo) containing only the terminal 191 bp of the AAV genomecan integrate normally, in a rescuable form, in the absence of AAV geneexpression. As discussed above, the AAV/neo stocks contained nodetectable helper or wild-type-like viruses. The recombinant viral DNA'swere judged to be integrated into the cellular genome of drug-resistantcell lines since no viral DNA was detected in preparations oflow-molecular-weight DNA, viral sequences were present inhigh-molecular-weight DNA, and DNA fragments of sizes consistent withvirus/host DNA junctions were observed upon DNA blot analysis (FIG. 5B).Two observations indicated that the recombinant viral DNA's wereintegrated normally. First, the viral sequences in a substantial portion(7/17) of the cell lines tested could be rescued by infection with amixture of adenovirus and AAV (FIG. 4). Second, all of the rescuablecell lines contained head-to-tail concatemers of the recombinant viralgenome. Head-to-tail concatemers have been described for a variety ofrescuable AAV inserts (Cheung et al., 1980, J. Viral. 33:739-748;Laughlin et al., 1986, J. Viral. 60:515-524; McLaughlin et al., 1988, J.Viral. 62:1963-1973). As yet it is not clear how they arise sincedimerized molecules produced during normal AAV DNA replication arehead-to-head or tail-to-tail repeats (Berns, 1984, The Parvoviruses,supra). Our results demonstrate that the mechanism by which head-to-tailconcatemers arise does not require AAV gene expression.

The present experiments rule out a requirement for AAV gene expressionwithin an infected cell for normal integration. Although it is clearthat integration has occurred in the complete absence of rep functions,we cannot rule out a possible direct role in the process for one or morecapsid proteins that enter the cell as structural elements of thevirion.

6.3.3. High Efficiency Transduction with Recombinant Viruses

The efficiency with which G418 resistance was transduced by the AAV/neostock was only slightly enhanced by addition of wild-type AAV to theinfecting mixture (at most, 4-fold, Table 2). In fact, high levels ofadded AAV reduced the efficiency of transduction (Table 2). Functionsencoded by the left half of the AAV chromosome (rep functions) have beenshown to negatively regulate AAV gene expression under certainconditions (Labow et al., 1986, J. Viral. 60:251-258; Tratshen et al.,1986, Mol. Cell. Biol. 6:2884-2894) and to interfere with expression ofgenes regulated by non-AAV control elements, including the SV40 earlypromoter (Labow et al., 1987, Mol. Cell. Biol. 7:1320-1325). McLaughlinet al. (1988, supra) have recently suggested rep gene products mightinterfere with efficient expression of non-viral marker genes inrecombinant AAV genomes. Our results are consistent with this view. Infact, we were able to achieve a very high frequency of transduction(about 70%) by infecting with AAV/neo at a multiplicity of 1000particles/cell in the absence of helper virus (Table III).

In general, it is difficult to compare the efficiency of transductionachieved with AAV/neo virus stocks to efficiencies reported by others.Earlier experiments (Hermonat and Muzyczka, 1984, Proc. Natl. Acad. Sci.U.S.A. 81:6466-6470; Lebkowski et al., 1988, Mol. Cell. Biol.8:3988-3996; McLaughlin et al., 1988, J. Viral. 62:1963-1973; Tratschinet al., 1985, Mol. Cell. Biol. 5:3251-3260) included helper virus in thetransduction inoculum, employed different virus constructs and utilizeda variety of protocols to select drug resistant cell clones. Further, avariety of methods for quantification of virus particles was employed.Nevertheless, in general, 1 to 3% of cells have been successfullytransduced to drug resistance, a frequency considerably lower than thatobtained by high multiplicity infection with AAV/neo virions in theabsence of helper AAV (Table III).

7. DEPOSIT OF MICROORGANISMS

The following microorganisms containing the indicated recombinantplasmids were deposited with the American Type Culture Collection inRockville, Md. Plasmid Host ATCC Accession no. pAAV/Ad * pAAV/neo *

The present invention is not to be limited in scope by the viruses andrecombinant DNA constructs exemplified or deposited microorganisms whichare intended as but single illustrations of one aspect of the invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

1-57. (canceled)
 58. A method for producing an adeno-associated viruspackaging cell line, comprising: a) transfecting cells permissive foradeno-associated virus replication with a helper adeno-associated virusDNA which provides viral functions sufficient for the replication andpackaging into infectious virions of recombinant adeno-associated virus,and which comprises a nucleotide sequence not found in wild-typeadeno-associated virus which promotes expression of adeno-associatedvirus genes which results in the viral functions provided, but whichlacks adeno-associated virus terminal repeat sequence; and b) selectingfor stably transfected cells which express said helper adeno-associatedvirus DNA.
 59. The method of claim 58, wherein said helperadeno-associated virus DNA comprises adeno-associated virus derivedcis-acting elements that control the expression of gene products. 60.The method of claim 58, wherein said helper adeno-associated virus DNAcomprises non-adeno-associated virus derived cis-acting elements thatcontrol the expression of gene products.
 61. The method of claim 58,wherein said helper adeno-associated virus DNA comprises induciblepromoter/enhancer elements that control the expression of gene products.62. The method of claim 58, wherein said helper adeno-associated virusDNA comprises adeno-associated virus genes located between aboutnucleotide 195 and about nucleotide
 4480. 63. An adeno-associated viruspackaging cell line produced using the method of claim
 58. 64. Anadeno-associated virus packaging cell line produced using the method ofclaim
 59. 65. An adeno-associated virus packaging cell line producedusing the method of claim
 60. 66. An adeno-associated virus packagingcell line produced using the method of claim
 61. 67. An adeno-associatedvirus packaging cell line produced using the method of claim
 62. 68. Amethod for producing a helper-free stock of recombinant adeno-associatedvirus comprising: a) transfecting an adeno-associated virus packagingcell line produced by the method of claim 58, with a recombinantadeno-associated virus vector which contains a foreign DNA sequence andwhich can be incorporated into an infectious virion, in the presence ofhelper virus infection; and b) collecting virions produced.
 69. Themethod of claim 68, wherein said helper virus is adenovirus or herpesvirus.
 70. The method of claim 68, wherein said recombinantadeno-associated vector comprises up to 195 base pairs of theadeno-associated virus terminal repeated sequence, a foreign DNAsequence, and shares no adeno-associated virus sequence with the helperadeno-associated virus DNA.
 71. The method of claim 68, wherein thehelper adeno-associated virus DNA comprises adeno-associated virusderived cis-acting elements that control the expression of geneproducts.
 72. The method of claim 68, wherein the helperadeno-associated virus DNA comprises non-adeno-associated virus derivedcis-acting elements that control the expression of gene products. 73.The method of claim 68, wherein the helper adeno-associated virus DNAcomprises inducible promoter/enhancer elements that control theexpression of gene products.
 74. The method of claim 68, wherein thehelper adeno-associated virus DNA comprises adeno-associated virus geneslocated between about nucleotide 195 and about nucleotide 4480.